FACS Analysis of Yeast Cultures

FACS analysis

Materials

Method

• Spin down 1-2 ml of culture at 5⋅10⁶ cell/ml;
• Resuspend the pellet in 1 ml of filtered 70% ethanol @ -20°C;
• Fix at 4°C at least two hours or overnight;
• Spin down and resuspend in 500 μl of 1 mg/ml RNase A obtained by diluting the 10X stock 1:10 in 50mM Tris-Cl pH 7.5;
• Incubate 2 hours @ 37°C. Overnight is also possible;
• Spin down and resuspend in 500 µl 50mM Tris-Cl pH 7.5 supplemented with 1 mg/ml Proteinase K;
• Incubate 2 hours at 42°C or 1 hour @ 50°C;
• Spin down 3′ @ 4°C and resuspend in 500 μl of FACS Buffer;
• Transfer 100 μl of cell suspension in a FACS tube containing 1ml of either”
• Sytox diluted 1:5000 in 50mM Tris-Cl pH 7.5
• 2 mg/ml Propidium Iodide, diluted 1:1000 in 50mM Tris-Cl pH 7.5
• Sonicate each sample 4 seconds and analyse.